It is the mission of the Flow Cytometry Core Facility to provide high quality instrumentation, scientific and technical expertise when consulting on experimental design, instrument training and assistance with data analysis. State-of-the-art instrumentation has been selected to ensure that NCI investigators have access to the most advanced flow cytometry cell analysis, cell sorting and data analysis available. Flow cytometry instrument training is conducted on one of the four flow cytometry cell analyzers in the Core, the BD FACSCalibur, BD FACSCanto II, BD LSR II or BD LSRFortessa. New users are instructed to review instrument and software training tutorials before attending the hands on training. After completion of the tutorials they attend a 4 hour training session that includes startup and shut down procedures for the cytometers, setting up the experiment, basic instrument troubleshooting, and analyzing and exporting the data to a shared NCI network for data archiving. The Core staff will provide follow-up assistance for performing the next several experiments and assist the new user in developing confidence in using this technology correctly. Assistance in selecting and optimizing the appropriate application and preparing a data presentation for laboratory meetings or publications is available. The flow cytometry cell analyzers are available to trained users on a 24/7 basis. Training on the BD FACSAria II cell sorter is offered to select individuals. When an investigator requires sorting to be done frequently, especially outside of normal working hours and has mastered the BD FACS DiVa software on the flow cytometry cell analyzers, they are considered as prospective candidates for cell sorting training. The major focus of NCI investigators using the Flow Cytometry Core Facility is to identify and study cancer cells. Many of the NCI investigators use flow cytometry and fluorescence-activated cell sorting to identify and sort cancer stem cells by membrane antigen expression using monoclonal antibodies or with a functional assay involving active membrane substrate transport. Investigators in CCBB, MBTL, LEC, LHC, and LCBG are studying cancer stem cells from breast, ovarian, hepatic, thyroid, pancreatic and lung carcinomas. The BD FACSCalibur, BD FACSCanto II, BD LSRII, and BD LSRFortessa flow cytometey cell analyzers, and two BD FACSAria II, and BC MoFlo Astrios EQ cell sorters are used for these experiments. Transfection of cells with genes expressing fluorescent reporters is a technique used by the majority of laboratories using the facility. The FCCF cell analyzers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with green, yellow, blue or red fluorescent proteins or with combinations of these fluorescent reporters. Sorted transfected cells are used to prepare protein, DNA, and RNA that can be used in Western blotting and microarrays. Sorted cells are also used to determine effects of siRNA, to look at signaling proteins, or may be further passaged to create stable cell lines. Fluorescent reporter proteins may also be linked to luciferase. Tumor cell lines have been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of expression. These cell lines have then been used to establish tumors in mice and to image metastasis. Flow cytometry is also frequently used for looking at cell growth (cell cycle analysis) and mechanisms of cell death (apoptosis) to examine actions of cancer drugs. Appointments to request instrument training and to use the analyzers are scheduled through the FCCF website https://labshare.nih.gov/nci/ccr-facs. The cell sorters are scheduled after the NCI, CCR, LGI Flow Cytometry Core Facility Biosafety Questionnaire and Sort Request form has been submitted and reviewed to determine the biosafety level required to perform the requested sort. This form submission is required from allinvestigators before a sort can be scheduled. The FCCF is compliant with the NIH Policy for Biosafety of Cell Sorters (July 28, 2012). Information about the sample source and potentially infectious agents is critical to ensure proper biosafety risk assessment. According to the NIH Policy for Biosafety of Cell Sorters (July 28, 2012), sorting of human cell lines, human cells and 2nd or 3rd generation Lentivirus in human cells require the sort to be performed at a BSL-2 with Enhanced Precautions biosafety level. The Beckman Coulter MoFlo Astrios cell sorter with SterilGARD Class II Biosafety Cabinet and an onboard HEPA filtration system provides safe, effective aerosol evacuation. The BD FACSAria Fusion cell sorter will provide the same level of safety when sorting samples of this type. A Class II type A2 Biosafety Cabinet is integrated into the instrument to ensure that aerosols are always contained and evacuated into the HEPA filters. Both cell sorters will be housed in the newly renovated space that is designated for sorts requiring BSL-2 with Enhanced Precautions. In addition to providing superior biosafety protection the BC MoFlo Astrios and BD FACSAria Fusion each have 5 lasers and 18 detectors capable of sorting cells stained with various combinations of fluorochromes. This year the FCCF instituted a 2015 Fee for Service Policy which charged Principal Investigators $2,000 annually when using the facility 3 or more times. After a sort, an instrument training session or assistance with an experiment, an additional use of the facility is allowed before the Investigator's budget is charged $2,000. This one time charge permits all personnel in said investigator's laboratory unlimited access to the resources of the Flow Cytometry Core Facility for the entire year. In 2015 the FCCF charged 58 Principal Investigators this fee. Investigators trained to operate the flow cytometry cell analyzers or the cell sorters have full access to the instruments 24/7. Training these individuals allow for more efficient use of the Core resources outside of normal working hours. The Flow Cytometry Core Facility has provided services to investigators from 11 Branches, 21 Laboratories and 3 Institutes. Because of the Core's expertise and specialized equipment not readily available in other institutes, we have provided both sorting and training to scientists outside of NCI.